The goal of our research is to elucidate the mechanism of activation and modulation of the G protein- gated K+ channel (KACh channel/GIRK) which regulates heart rate and synaptic transmission. Recent studies have shown that interaction of the KACh channel with phosphatidylinositol-4,5-bisphosphate (PIP2) in the membrane is critical, as this allows other gating molecules (beta gamma, Na+) to work. Betagamma is the most effective gating molecule as it produces the greatest activation. We recently discovered the existence of an inhibitory lipid substance in the cytoplasm of atria and brain that plays a crucial role in KACh channel function. In the absence of the endogenous lipid inhibitor (excised patch), the KACh channel shifts to a "high open probability" mode with long- lived openings. In the presence of the inhibitor (cytoplasm), the KACh channel activity is much lower showing only short-lived openings, similar to the KACh channel state observed in cell-attached patches (intact cells). Using electrophysiological and molecular biological methods, we propose to identify the endogenous inhibitor and study its physiological role in KACh channel function by selectively removing it from the cell (specific aim number 1). Using chimeric channels (GIRK/IRK) and PIP2 antibody, we will study the molecular basis for the inhibitory effect on the KACh channel activity. This will be done by testing the hypothesis that the inhibitor blocks PIP2-KACh channel interaction and thus reduces the effectiveness of gating molecules such as betagamma and Na+ (specific aim number 2). As changes in KACh channel kinetics produced by the cytoplasmic inhibitor are similar to those observed during the fast desensitization, we will test the hypothesis that the putative inhibitor mediates the fast desensitization of the agonist-induced KACh current (specific air number 3). These studies should reveal novel signaling pathways involved in agonist-induced activation and desensitization of the KACh channel, and help better understand the control of heart rate.